Tenascin-C isoform as marker for neoplasias

ABSTRACT

Diagnostic method for the identification of human neoplasias based on the determination of the CTN-C isoform of TN-C, fragments antibodies and their conjugates used in said method, and their therapeutic use.

FIELD OF THE INVENTION

The present invention refers to a diagnostic method allowing a highlyspecific identification of human neoplasias by means of fragments ofrecombinant human antibody, and to said fragments and conjugatesthereof. The invention also refers to the use of fragments andconjugates thereof in the preparation of therapeutically usefulformulations.

STATE OF THE ART

During neoplastic growth, the extracellular matrix (hereinafter referredto as ECM) of normal tissues, where tumour growth takes place, isremodelled by processes of proteolytic degradation and synthesis of newcomponents.

ECM components of tumors differ from those of normal tissues, in termsof quantity and quality, and contribute to the creation of conditionsfavoring tumor growth and development, among which is angiogenesis,which plays a major role in neoplastic development.

Tenascin-C (hereinafter referred to as TN-C) is a glycoproteinconsisting of six similar subunits linked by disulphide bonds. It iscoded for by a single gene and its expression is regulated by a singlepromoter.

Through a mechanism known as alternative splicing, 9 proteic domains,homologous to the “type III fibronectin domains” (hereinafter referredto as FNIII) can be either included in or omitted from the mRNA of humanTN-C, giving rise to various proteic isoforms.

It is also known that TN-C isoforms are abundantly present in normaladult tissues and that TN-C isoforms including most or all 9 domainsmentioned above are very widely expressed in neoplastic tissues.

The reagents suitable for identifying TN-C isoforms available to dateare murine monoclonal antibodies that, as such, are not appropriate tobe used in man (e.g. immunoscintigraphy); furthermore, they reactindiscriminately with the isoforms present in neoplastic and healthytissues.

Therefore, it is clear that it would be of major importance to develop amethod and reagents allowing the identification of the isoforms presentin neoplastic tissues alone. This, in fact, would permit a highlyprecise and specific diagnosis, while making it possible for a drug oranother effector to reach the tumour only, for therapeutic purposes.

DESCRIPTION OF THE FIGURES

FIG. 1A: Structural model (in domains) of a subunit of human TN-C. Ovaland square symbols represent EGF-like and FN-like repeats, respectively.The amino terminal and the fibrinogen-like terminal COOH knob are alsoshown. FN-like repeats from A1 to D, whose expression is regulated bypre-mRNA alternative splicing, are shaded. The top part of the figurealso shows the TN-C-β-galactosidase fusion proteins or recombinantproteins used. Arrows indicate the position of the epitopes of eachrecombinant or monoclonal antibody. A indicates continuity.

FIG. 1B: Electrophoresis in Sodium Dodecyl Sulphate (4–18% SDS. PAGE) of‘long’ recombinant protein TN-C (containing domains A1 to D) and ‘short’TN-C (not containing domains A1 to D), stained with Coomassie blue, andimmunoblots stained using scFv TN11 and TN12.

FIG. 1C: Immunoblots of various fusion and recombinant proteins (A),stained with scFv TN11 and TN12. The values reported on the leftindicate the molecular mass (in kilodaltons) of standards.

FIG. 2: Northern blots of poly(A)-rich RNA obtained from adult humantissues of (1) heart, (2) brain, (3) placenta, (4) lung, (5) liver, (6)skeletal muscle, (7) kidney, and (8) pancreas, and from fetal humantissues of (1) brain, (2) lung, (3) liver and (4) kidney, obtained usingthe cDNA probe described in the text, which is specific for the cTN-Cisoform, the HT11 probe recognising all TN-C isoforms, and the cDNA ofhuman G3PDH for blots normalisation. Numbers on the left representmeasurements of standards (in kb).

FIG. 3: Immunohistochemical analysis of glioblastoma sections with scFvTN11 (A and B) and with double staining obtained with scFv TN11 (red)and mAb KI67 (brown) (C, E, F, and G); section of cerebral metastasis ofpulmonary carcinoma stained with scFv TN11 (D). Bar=10μ.

FIG. 4: Immunohistochemical analysis of serial sections of invasiveductal carcinoma of the breast using scFv TN12 (A and C) and scFv TN11(B and D) and serial sections of meningioma stained with scFv TN12 (E)and scFv TN11 (F). Bar=10μ.

FIG. 5: Demonstration by Southern blot of the specificity of the cRNAprobe used for in situ hybridisation experiments. Bottom: staining ofagarose gel with ethidium bromide. 1: TNFNALL (which comprises the DNAof human TN-C from domain 2, type III to domain 7, type III, includingthe domains subjected to splicing); 2: TNFN1–8 (the same as the TNFNALLsequence, but without the domains subjected to splicing); 3: allTNEGF-like domains; 4: domain D, type III; 5: domain C, type III; 6:domain 1, type III; 7: TNEGF-like domains from 8 to 10; 8: standard.

Top: Southern blot of the same fragments as described above, hybridisedwith the DIG-labelled probe. Numbers on the right are measured in kb.

FIG. 6: Two magnifications of in situ hybridisation experiments on humanglioblastoma cryostat sections, using the DIG-labelled cRNA probe ofdomain C. The positive signal is visible only in some tumour cellshaving large nucleus.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that human TN-C isoforms containing domain C(hereinafter referred to as cTN-C) are greatly expressed in vascularstructures and in proximity of high grade astrocytoma proliferatingcells (grade III) and glioblastoma. The isoforms in question are alsowidely expressed in vascular structures of pulmonary human neoplasia,whereas they are not detected in any normal adult human tissue.

Therefore, the present invention refers to a method for theidentification of neoplastic tissues in vivo and in vitro, based on thedetermination of the presence of the cTN-C isoforms of TN-C.

The invention also refers to ligands capable of recognising cTN-C andconjugates thereof.

The term ‘ligands’ is used herein to mean antibodies or fragmentsthereof or any other molecule capable of recognising and binding itselfto cTN-C.

In particular, according to the present invention, ‘ligands’ arefragments of recombinant human antibodies and more particularly scFvfragments. In fact, compared with conventional immunoglobulins, smallfragments of human antibodies, as the scFv fragments are, do notaccumulate in the liver, are not immunogenic and exhibit a betterpenetration into tissues.

According to the invention, useful conjugates can be obtained by knowntechniques by biochemical or genetic conjugation of the ligand for cTN-Cto the molecules suitable for the fixed diagnostic and/or therapeuticpurpose. Appropriate molecules for the conjugation with the ligand maybe, e.g., radioisotopes, fluorescent substances, cytokines, toxins,photosensitizers, thrombogenetic agents, etc.

To construct the conjugates according to the invention it is possible touse, e.g., the ligand described above as well as peptides or othernon-proteic molecules. Particularly interesting, according to theinvention, is the ligand represented by the recombinant human antibodyscFv, whose sequence is reported in Table 1 (SEQ ID NO. 1) hereinafterreferred to as TN11.

TABLE 1 TN11 sequence deduced from the DNA sequence1/1                                     31/11CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG TCC TCG GTG AAG GTCQ   V   Q   L   V   Q   S   G   A   E   V   K   K   P   G   S   S   V   K   V61/21                                   91/31TCC TGC AAG GCT TCT GGA GGC ACC TTC AGC AGC TAT GCT ATC AGC TGG GTG CGA CAG GCCS   C   K   A   S   G   G   T   F   S   S   Y   A   I   S   W   V   R   Q   A121/41                                  151/51CCT GGA CAA GGG CTT GAG TGG ATG GGA GGG ATC ATC CCT ATC TTT GGT AGA GCA AAC TACP   G   Q   G   L   E   W   M   G   G   I   I   P   I   F   G   T   A   N   Y181/61                                  211/71GCA CAG AAG TTC CAG GGC AGA GTC ACG ATT ACC GCG GAC GAA TCC ACG AGC ACA GCC TACA   Q   K   F   Q   G   R   V   T   I   T   A   D   E   S   T   S   T   A   Y241/81                                  271/91ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG TAT TAC TGT GCG AGA TCG AGAM   E   L   S   S   L   R   S   E   D   T   A   V   Y   Y   C   A   R   S   R301/101                                 331/111CGT ATT ACG ATT TTT GGA GGA GGT GCT TTC GAT ATC TGG GGC CGA GGC ACC ATG GTC ACCR   I   T   I   F   G   G   G   A   F   D   I   W   G   R   G   T   M   V   T361/121                                 391/131GTC TCT TCA GGT GGG GGC GGT TCA GGC GGA GGT GGC AGC GGC GGT GGC GGA TCG CAG TCCV   S   S   G   G   G   G   S   G   G   G   G   S   G   G   G   G   S   Q   S421/141                                 451/151GTG CTG ACT CAG CCT GCC TCC GTG TCT GGG TCT CCT GGA CAG TCG ATC ACC ATC TCG TGCV   L   T   Q   P   A   S   V   S   G   S   P   G   Q   S   I   T   I   S   C481/161                                 511/171ACT GGA ACC AGC AGT GAT GTT GGT GGT TAT AAC TAT GTC TCC TGG TAC CAA CAA CAC CCAT   G   T   S   S   D   V   G   G   Y   N   Y   V   S   W   Y   Q   Q   H   P541/181                                 571/191GGC AAA GCC CCC AAA CTC ATG ATT TAT GAG GGC AGT AAG CGG CCC TCA GGG GTT TCT AATG   K   A   P   K   L   M   I   Y   E   G   S   K   R   P   S   G   V   S   N601/201                                 631/211CGC TTC TCT GGC TCC AAG TCT GGC AAC ACG GCC TCC CTG ACA ATC TCT GGG CTC CAG GCTR   F   S   G   S   K   S   G   N   T   A   S   L   T   I   S   G   L   Q   A661/221                                 691/231GAG GAC GAG GCT GAT TAT TAC TGC AGC TCA TAT ACA ACC AGG AGC ACT CGA GTT TTC GGCE   D   E   A   D   Y   Y   C   S   S   Y   T   T   R   S   T   R   V   F   G721/241 GGA GGG ACC AAG CTG ACC GTC CTA GGTG   G   T   K   L   T   V   L   GExperimental PartIsolation of Antibody Fragments Against the ‘Long’ TN-C Isoform

A phage display library of human scFv was selected using, as an antigen,the ‘long’ TN-C variant including all FNIII domains subjected toalternative splicing. Culture media of bacterial colonies obtained fromsaid selection were analysed by the ELISA technique using, as antigens,TN-C variants with all FNIII domains (‘long’) or with no FNIII domain(‘short’) subjected to alternative splicing.

This investigation allowed the identification of a clone producingspecific antibodies for the TN-C ‘long’ form. From the supernatant ofthe bacterial culture of said clone, denominated TN11, scFv was purifiedby column immunochromatography on Sepharose conjugated with recombinantfragment A-D (containing all FNIII domains subjected to alternativesplicing).

TN11 was further characterised by the immunoblotting technique, whichallowed the evaluation of the specific reaction with ‘long’ and ‘short’TN-Cs and with various recombinant and fusion proteins (TN A-D, TN B-D,TN C, TN B, λTN27 and λTNBC) containing various domains of human TN-C(FIG. 1 A). Said technique provided evidence that TN 11 not onlyrecognised the TN-C ‘long’ form (as already found by the ELISA techniqueduring selection phases), but also reacted specifically with all proteicfragments containing domain C (FIG. 1 B, C). This was a proof that theepitope recognised by TN11 is located inside domain C of TN-C. Similarinvestigations proved that TN11 does not react with the TN-C purifiedfrom the culture medium of normal human fibroblasts, because the TN-Cproduced by said cells does not contain domain C.

RT-PCR experiments were conducted on total RNA extracted from culturesof normal human fibroblasts (GM-6114, ATCC, Rockville, Md., USA), fromcells derived from human melanoma (SKMEL-28, ATCC, Rockville, Md., USA)and from tissue samples of human glioblastoma and meningioma, using thefollowing primers:

5′ GCTACCCCCTAGTACTGATTTTATTGTCTA (from base. 4542 to 4571 of human TN-Csequence) (SEQ ID NO. 3),

5′ TTTCCAGTGGCTCAGACTGC (complementary sequence, from base 5028 to base5047) (SEQ ID NO. 4),

5′CTGGTCTGAGTCTTGGTTCCGTCC (complementary sequence, from base 5322 tobase 5345) (SEQ ID NO. 5).

RT-PCR experiments evidenced that domain C of TN-C, which is absent incells GM-6114 and SKMEL-28 as well as in meningiomas, is present in themRNA of TN-C, purified from human glioblastoma fragments.

Northern blotting analyses conducted using mRNA from normal humantissues, adult and embryonic respectively, and a cDNA probe containing270 bases (4630 to 4899) of the human TN-C sequence, demonstrated thatthe mRNA of this domain is expressed only in fetal tissues (brain,liver, kidney) and is absent in the mRNA of adult tissues (FIG. 2).

Binding bond affinity of purified antibody TN 11 to ‘long’ TN wasdetermined by interaction analysis using BIAcore. The dissociationconstant was found to be 1.3×10−10.

The immunohistochemical analysis conducted using TN11, which is specificfor domain C of TN-C, confirmed that domain C cannot be found in normaladult tissues. Conversely, there is a large presence of total TN-C(evidenced by the reaction with monoclonal antibody BC-4, specific forall human TN-C isoforms, since it recognises an epitope of the humanTN-C molecule constant zone.

It was also found that almost all glioblastomas investigated expressvery high levels of domain C, with 14 cases of tumour out of the 15highly positive ones (Table 2 and FIG. 3). In particular, the presenceof this TN-C isoform was mainly identified in proximity of vascularstructures in areas with high cellular proliferation activity, in thestroma of tumour cell nests (FIGS. 3 A, B, C, E, and G), and inproliferating cells (FIG. 3 F). Conversely, no positive reaction wasobtained in other tumours of the brain, excepting 2 meningiomas out ofthe 23 that were weakly positive in proximity of vascular structuresonly (Table 2 and FIG. 4).

A large presence of cTN-C in pulmonary neoplasm sections, especially inproximity of vascular structures, was also observed.

In situ hybridisations of glioblastoma cryostat sections (FIGS. 6 A andB) were conducted using DIG-labelled cRNA probes, specific for cTN-C(FIG. 5). The results obtained prove that the cTN-C isoform is producedby tumour cells, but not by all tumour cells.

cTN-C expression in vascular structures was also demonstrated in anexperimental model of human melanoma, in nude mice (using SK-MEL-28cells). Human-melanoma-carrier nude mice were injected withradio-labelled scFv TN11, which proved the antibody specificaccumulation in tumour vascular structures only.

To conclude, the determination of the presence of the cTN-C isoform ofTN-C is a valid method for the diagnosis of various types of tumours.Furthermore, the presence of said isoform in neoplastic tissues may beuseful also for therapeutic purposes.

TABLE 2 Reactivity of scFv TN11 and TN12 with primary tumours ofdifferent histotype No. of positive/No. of tested cases Tumour TN11 TN12Glioblastoma and anaplastic astrocytoma 15/16 16/16 Astrocytoma Grade II0/5 5/5 Pilocytac astrocytoma 0/2 2/2 Neurinoma 1/1 1/1 Ependymoma 0/11/1 Oligodendroglioma (1) 1/2 2/2 Meningioma (2)  1/23 23/23 Cerebralmetastasis (3)  7/15 15/15 Breast adenocarcinoma (4)  3/27 27/27 Stomachadenocarcinoma 0/2 2/2 Lung carcinoma 19/24 24/24 All tumours werehighly positive with scFv TN12, which identifies all TN-C isoforms. scFvTN11 identifies the TN-C isoform containing domain C only. (1) Thepositive case exhibited positivity only in some vascular structures. (2)The positive case was a transition meningioma and exhibited positivityonly in some vascular structures. (3) Out of the 7 positive cases, 3exhibited positivity in the connective tissue and in some vascularstructures, and 3 exhibited positivity in some vascular structures only.(4) In 3 positive cases, staining could be slightly evidenced.

All tumours were highly positive with scFv TN12, which identifies allTN-C isoforms. scFv TN11 identifies the TN-C isoform containing domain Conly.

(1) The positive case exhibited positivity only in some vascularstructures.

(2) The positive case was a transition meningioma and exhibitedpositivity only in some vascular structures.

(3) Out of the 7 positive cases, 3 exhibited positivity in theconnective tissue and in some vascular structures, and 3 exhibitedpositivity in some vascular structures only.

(4) In 3 positive cases, staining could be slightly evidenced.

1. A monoclonal antibody or antigen binding fragment thereof capable ofidentifying the human TN-C isoforms containing domain C (cTN-C) whereinsaid antibody or antigen binding fragment thereof binds to domain C inisoforms of TN-C expressed in cancer cells and/or vascular structures ofmalignant tumors and not to isofoms of TN-C expressed in normal adulthuman tissue.
 2. The antibody or antigen binding fragment thereof asclaimed in claim 1, wherein said antigen binding fragment is scFv, or anscFv fragment.
 3. The antigen binding fragment as claimed in claim 2wherein said antigen binding fragment has the amino acid sequence of SEQID NO.
 1. 4. A diagnostic reagent comprising a conjugate consisting of amonoclonal antibody as claimed in claim 1 and a molecule selected fromthe group consisting of radioisotopes, fluorescent substances, andphotosensitizers, allowing its use for diagnostic purposes.
 5. A methodfor the preparation a reagent diagnostic for malignancy/cancer for thedetermination of cTN-C isoforms of TN-C in tissues and/or biologicalfluids comprising admixing the antibody or the antigen binding fragmentthereof of claim 1 with a pharmaceutically acceptable carrier.
 6. Amethod for the preparation of a reagent for diagnosing malignancy/cancercomprising admixing the diagnostic reagent of claim 4 with apharmaceutically acceptable carrier wherein said diagnosing identifiescTN-C in tissues and or biological fluids by the binding of saiddiagnostic reagent to said cTN-C.
 7. A method for the preparation of acomposition comprising the antibody or antigen binding fragment thereofof claim 1 comprising admixing said antibody or antigen binding fragmentthereof with a pharmaceutically acceptable carrier.
 8. A conjugateconsisting of a monoclonal antibody or an antigen binding fragmentthereof as claimed in claim 1 and a molecule selected from the groupconsisting of radioisotopes, fluorescent substances, andphotosensitizers allowing its use for diagnostic purposes.
 9. Aconjugate consisting of a monoclonal antibody or an antigen bindingfragment thereof as claimed in claim 2 and a molecule selected from thegroup consisting of radioisotopes, fluorescent substances, andphotosensitizers allowing its use for diagnostic purposes.
 10. Aconjugate consisting of a monoclonal antibody or an antigen bindingfragment thereof as claimed in claim 3 and a molecule selected from thegroup consisting of radioisotopes, fluorescent substances, andphotosensitizers, allowing its use for diagnostic purposes.
 11. A methodfor the preparation of a reagent for diagnosing malignancy/cancercomprising admixing the conjugate of claim 9 with a pharmaceuticallyacceptable carrier wherein the diagnosing identifies cTN-C in tissuesand/or biological fluids by the binding of said diagnostic reagent tosaid cTN-C.
 12. A method for the preparation of a diagnostic reagent fordiagnosing malignancy/cancer comprising admixing the conjugate of claim10 with a pharmaceutically acceptable carrier wherein the diagnosingidentifies cTN-C in tissues and/or biological fluids by the binding ofsaid diagnostic reagent to said cTN-C.